Fixing and drying cytological smears



3,389,052 FIXING AND DRYING CYTOLOGICAL SMEARS Theodore Ehrenreich, 400 E. 57th St., New York, N.Y. 10022, and Stase Kerpe Zadeikis, 85-48 148th St., Jamaica, N.Y. 11435 No Drawing. Continuation-impart of application Ser. No. 277,466, l t/lay 2, 1963, which is 'a continuation-in-part of application Ser. No. 810,532, May 4, 1959. This appiication Nov. 2, 1965, Ser. No. 506,124

16 Claims. (Cl. 167-845) This application is a continuation-in-part application of my copending application Ser. No. 277,466, filed May 2, 1963, now abandoned which in turn is a continuationin-part application of application Ser. No. 810,532, filed May 4, 1959, now abandoned.

This invention is that of a method of fixing and drying a cytological smear on a carrier, e.g., a microscope slide, without contaminating the smear, and to enable it to keep without spoiling and be transported conveniently even by mail, and subsequently to be subjected directly to an aqueous staining solution without need for any intervening preparatory treatment.

This invention includes also fixing and drying compositions that enable accomplishing the foregoing. These compositions consist essentially of (a) from about 79 to about 89 parts of a compatible Freon aerosol propellant inert to the smear, (b) from about 0.25 to about 0.92 part of a film-forming mixture of polyethylene glycols soluble in a water-miscible lower alkanol solvent (containing from about 3 to about 10 percent of Water) and miscible with the propellant, and (c) from about 10 to 19.5 parts of a common solvent for the propellant and the polyethylene glycol mixture (b), which solvent is the aforesaid lower alkanol solvent and is also a dehydrating agent.

For over the past 20 years detection of certain ailments was attempted by the examination of the cellular debri eliminated in a body cavity and picked up as cytological smears, for example, a vaginal smear in the search for cancer. As only very few diagnostic physicians are equipped to examine such smears directly, they have to be transmitted to laboratories equipped for the microscopic examination of the cell structures picked up in such smears.

During that period, a number of dilferent methods were considered for preparing dry fixed smears on glass slides for cytological examination. Their aim was to achieve (a) good preservation of the cells in the fixing and drying of the smears on the glass slides which are used for microscopic examination, and (b) a convenient and safe means of shipping these dry, fixed smears to the laboratory.

The original one of these methods is described by Papanicolaou in his article entitled, A New Procedure for Staining Vaginal Smears, in Science (1942), volume 95, pages 438-439. In it, the glass slides bearing the freshly taken smears, were immersed in a bath of a mixture of equal parts of ethyl alcohol and ethyl ether, held in a bottle. This method, still widely used, gave good results in fixing the cells on the slides, but the bottles are bulky and breakable, and not suitable for mailing.

Ayre and Dakin, in Cervical Cytology Tests in Cancer Diagnosis; Glycerine Technique of Mailing, in Canad. MAJ. (1946) vol. 54, pages 489-491, suggested fixing the smears on the slides by immersing them in a bath of a mixture of ethyl alcohol and ethyl ether and then covering each smear on its slide with glycerine and covering that with a clean slide. That was cumbersome and inconvenient.

Sills, in Dry Method for Conserving and Transporting Cytological Smears, Correspondence, J.A.M.A. (1953), vol. 151, page 230, described a method of first fixing the United States Patent smears on glass slides by immersing them in the ethyl alcohol and ethyl ether mixture, then removing the slides from that bath, evaporating the entrained mixture from the glass slides and smears, and then dipping them into melted polyethylene glycol known commercially as Carbowax 1540. This molten Carbowax. 1540 solidifies when the slide is removed from its molten bath, and forms a tough protective coat on the fixed smears.

In The Use of Acetone as a Fixative in Exfoliative Cytological Studies (1957) Amer. J. Obs. Gyn., V 1. 73, pages 437-439, Sagi andtMackenzie describe a method of fixing smears on the glass slides by immersing them for 20 to 30 minutes in acetone, and permitting the slides to dry.

Nieburgs in Simplified Cytology for Ofiice Practice, in Am. J. Clin. Path. (vol. 27, pages 546-550, May 1957) suggested mailing non-fixed, but dry smears on glass slides and then rehydrating the dry smears on the slides in the laboratory before staining the rehydrated smears. He claimed that the non-fixed smears remain in satisfactory condition for a period as long as two weeks. However, when received in the laboratory, the slides bearing these dry smears must be immersed in a rehydrating fluid for from one to five minutes, and then must be rinsed in equal parts of a mixture of ethanol and ethyl ether. The smears on the slides then are fixed for ten minutes in another bath of the mixture of 95 ethanol and ethyl ether. The slides then are treated with ethyl alcohol in successively different concentrations with water, of 95%, 70%, 35% ethanol respectively.

Later, in his Simple Method for Protecting Fresh Smears from Drying and Deterioration During Mailing, in J.A.M.A., vol. 164, pages 1330-l331 (1957), Papanicolaou suggested covering the wet smear with a solution of a synthetic mounting resin (Diaphane) dissolved in alcohol, which solution dries in 20 to 30 minutes when applied to the wet smear on its slide, In the laboratory, the resulting dry resin coating must be removed from each dry slide, before staining the smear, by immersing it on its slide for 20 minutes in a bath of mixed ethyl alcohol and ethyl ether in equal parts.

In Mailing of Fixed and Dried Cytological Smears, Correspondence, J.A.M.A. vol. (December 1957), pages 2219-2220, Sills and Garrett describe their method using a solution of the polyethylene glycol Carbowax 1540 in a mixture of equal parts of ethyl alcohol and ethyl ether. The slides bearing the freshly taken smears are immersed for 30 to 60 minutes in this mixture solution. The slides then are removed and dried. As a result of this treatment, the cytological smears are fixed and provided with a coating of this polyethylene glycol Carbowax 1540.

The various shortcomings and disadvantages, for example, time consumption, cost, handling of dangerous solvents such as ethyl ether and acetone, extra steps, shipping hazards, and others in these earlier methods are avoided by drying and fixing cytological smears according to the method of this invention, which involves using the aerosol compositions of the invention.

A feature of the invention is the ability to fix and dry 2. cytological smear on a glass slide carrying it, simply by subjecting the smear on its slide merely to contact with an aerosol spray dispersion of an aerosol composition of the invention to coat the smear, and allowing it and the resulting spray deposit over it to dry in the room atmosphere.

Another feature of the invention is that the smear and the aerosol spray deposit coating on it are adequately fixed and dried safely within about 5 to 7 minutes and only under the ambient atmospheric conditions. This is an unusually short time.

A further feature of the invention is that the thus quickly dried smear and its momentarily applied aerosol spray deposit leave the smear fixed and dried under a penetrating film-coating of a dry to the touch, generally translucent, firm and resistant to flow, waxy film of the original solid polyethylene glycol content of the aerosol composition.

Still another feature of the invention is that the thus dried and fixed smear with such dry to the touch, firm and flow resistant, waxy solid polyethylene glycol coating over it on the microscope slide is ready for transmis sion by mail protected only by means ordinarily used for preventing breakage of any such flat glass slide.

Yet a further feature of the invention is that such fixed and dried smear with its dry to the touch, firm and flow resistant, waxy solid polyethylene glycol coating over it is ready for staining simply by immersing the thus polyethylene glycol coated slide, after reaching the laboratory, into the ordinary aqueous hematoxylin staining solution. This dissolves oil the solid polyethylene glycol coating from over the slide and smear while during the same immersion the smear is stained in the same way as ordinarily occurs to a fixed smear in such hemotoxylin solution.

Considered generally, the method of the invention involves simply interposing an ordinary cytological smear, as borne on a usual carrier for it, in the finely divided spray dispersion emitted from the nozzle of an aerosol dispenser containing a composition of this invention, and at such distance from the nozzle to avoid disrupting the smear by excessive emission pressure, to cover the smear after one or two or so passes of the spray over it with a substantially complete coating with the aerosol dispersion.

Thereby, after allowing the lower alkanol mutual solvent (for the polyethylene glycol and Freon propellant) and the propellant to evaporate off within several minutes under the ambient atmospheric conditions, the smear remains fixed and dried under a penetrating coating of a dry to the touch, firm and flow resistant, waxy deposit of the originally dissolved solid polyethylene glycol content of the aerosol composition of the invention.

This waxy deposit is a penetrating, water-soluble, waxy film-coating which is non-brittle, and thus resistant to cracking and breaking during handling and transit. Its firmness and flow resistance maintains its form and shape at the various temperatures usually encountered before it is to be immersed in the staining solution.

It is refer-red to as penetrating because it does not lie as a merely flat-bottomed film on top of the smear thus dried by its evaporated solvent; but rather the waxy-filmcoating is infiltrated between and about the dried cells of the original smear. That is so because the aqueous alkanol solution of the film-forming constituent of the aerosol composition, when freshly spray-coated over the smear, commingles and mixes with the aqueous content of the original wet smear and so penetrates down between and around the particles of the initial ordinary dispersed solid content of the smear.

Considered broadly, the aerosol compositions of the invention have the property of being able to dry and fix cytological smears on the usual carriers for them, and consist essentially of (a) an aerosol propellant, (b) a film-forming mixture of polyethylene glycols, and (c) a water-miscible lower alkanol common solvent for the propellant and the film-forming mixture; each of which three has its individual properties and is used in its specific percentage range, respectively as follows:

(a) From about 79 to about 89 percent being the propellant which is a relatively non-toxic chloroand fluorosubstituted methane or ethane, having at most only one non-replaced hydrogen and a molecular weight of from about 103 toabout 204 and being non-reactive with the film-forming mixture and the mutual solvent and miscible with the latter;

(b) Under percent and from about 10 to about 19.5 percent of the water-miscible lower alkanol mutual solvent which also is a dehydrating agent, miscible with the propellant, and contains an amount of water up to about 10 percent of said alkanol sufficient to enable its dissolving the amount of the propylene glycol film-forming constituent used in the composition; and

(c) Under one percent and from about 0.25 to about 0.95 percent of a water-soluble film-forming mixture of polyethylene glycols, which is from stably dispersible to soluble in said lower alkanol solvent with the indicated range of water content and has such average molecular weight as to remain as a fixed, dry to the touch, nonbrittle, firm and flow resistant, waxy film-deposit after evaporation of the propellant, the mutual alkanol solvent and the contained water from a spray-deposited coating of said composition over a surface.

The applicable aerosol propellants include the halogen substituted methanes and ethanes, wherein both of the halogens chlorine and fluorine are linked to each carbon and with a total of at least three halogen atoms linked to the carbon in methane and each of the carbons in ethane. The chemical identity of a number of illustrative examples of these applicable aerosol propellants are for brevity of reference accompanied by their respective trademarks of one of the manufacturers. Thus, for example, illustrative applicable methanes are dichlorodifiuoromethane (Freon-l2), trichloromonofluoromethane (Freon1l), both of which have each of the carbon valences linked to a halogen, also dichloromonofluoromethane (Freon-21) and monochlorodilluoromethane (Freon-22). The latter two exemplify methanes having one non-replaced hydrogen.

In the applicable chloroand fiuoro-ethanes, each of the other valences of each of the two carbons is linked to one of these two halogens, for example, 1,2-dichlorotetrafiuoroethane (Freon-114"), and 1,1 dichloro-lfiuoro 2 chlorodifiuoroethane (Freonll3). Others deemed also effective have relatively similarly applicable boiling points and evaporation rates for use either alone or admixed with any one or more of the others of the same type.

So far as presently indicated, Freon-l2 may be used alone as the propellant, and at times so also may be Freon-I14 although the latter evaoprates more slowly than does Freonl2. Thus, for many of the fixing and drying compositions of the invention, it is more advantageous to employ suitable mixtures, often approaching about equal parts, of one of these propellants which evaporates more rapidly than most of the others admixed with one or more of the more slowly evaporating propellants. An advantageously efiective example is 40 parts of Freon-l2 with 60 parts of Freon-114. A good example includes equal parts of Freonl2 and Freon 11. The ratios, of course, can be varied to give a more effective vapor pressure to the final aerosol composition, for example, at from about 30 to about 75 pounds per square inch at about F. In some cases, for example, there may be used two parts of Freon 12 to one part of Freon-ll.

The applicable water-miscible lower alkanol mutual solvent can be any of isopropanol, ethanol, propanol and methanol, given in decreasing order of preference, or any suitable mixtures of any of them. Each of them can be used in its readily available cornmerical form, such as isopropyl alcohol containing from 9 to 10% of water, ethyl alcohol containing about 5% of water. Each of these alkanols is included in the complete composition so that any of these specific alkanols appear together with an amount, from about three to about ten percent, of water sufficient to enable dissolving the quantiy of the mixture of polyethylene glycols film-forming constituent of the specific complete composition.

Such alkanol constituent also is a dehydrating agent. It serves as such agent, after evaporation of the propellant leaves the waxy deposit of the fixing film-coating component of the entire composition therefrom over the cytological smear on its carrier, by evaporating from the deposited film of the polyethylene glycols mixture solution not only together with the water contained therein from the original complete composition as so applied but also with the water content of the original smear.

The constituent of the complete aerosol composition, which provides the ultimate Watensoluble, dry to the touch, non-brittle, firm and resistant to fiow, waxy filmcoating covering the dried smear is composed of a suitable Water-soluble mixture of one or more of (i) the ordinarily liquid polyethylene glycols with one or more of (ii) the ordinarily solid polyethylene glycols.

Such suitable mixture contains these respective Watersoluble glycols (i) and (ii) in such proportions to one another that the watensoluble solid film left after there evaporates off the alcohol and its accompanying water content from a liquid film of a solution of the mixture of these glycols (i) and (ii) in the water-miscible lower alkanol mutual solvent containing water to the extent of from about three to about ten percent of its alkanol content, has the required dry to the touch, non-brittle, firm and resistant to flow, waxy character.

The applicable starting polyethylene glycols conveniently can be identified by the trademark of one of their manufactures, wherein the numeral part of the trademark usually indicates the average molecular weight of the particular polyethylene glycol. Thus, the ordinarily liquid polyethylene glycols which can be used in preparing the mixture of such glycols to be included in the complete compositions are Carbowax 200 (av. mol. wt. range 190-210), Carbowax 300 (av. mol. Wt. 285-315), Carbowax 400 (av. lll'lOl. wt. 380-420), and Carbowax 600 (av. mol. Wt. 570-630). Then, the applicable ordinarily solid polyethylene glycols include Carbowax 1000" (av. mol. wt. 950-1050), Carbowax 1540 (av. mol. wt. 1300-1600), Carbowax 4000" (av. mol. wt. 3000-3700), Carbowax 6000 (av. mol. wt. 6000-7500), and Carbowax 20M (where M equals 1,000; estimated av. mol. wt. 15,000-20,000).

Also included among the ordinarily solid polyethylene glycols is Carbowax 1500, but its numeral does not indicate its average molecular Weight. It is a blend of equal parts of Carbowax 300 and Carbowax 1540, has an average molcular weight between 500 and 600, and is a bland, odorless solid with the consistency of lowmelting petrolatum.

Carbowax 1000 is a Waxy, white semi-solid melting between 98.6 and 104 F. Carbowax 1540 is a white solid of beeswax consistency and melts between 104 and 113 F. Each of Carbowax 4000, Carbowax 6000, and Carbowax 20M is a hard, translucent, waxy white solid resembling paraffin in appearance and texture, which softens at about 127.5 F. and so melts within several degrees higher. These various Carbowax polyethylene glycols are more fully described in the literature, e.g., a printed booklet copyrighted and published in 1958 by Union Carbide Chemicals Corporation.

The polyethylene glycol mixture may include, for example, by weight, of about six parts of polyethylene glycol having an average molecular weight from 3,000 to 3,700, to four parts of polyethylene glycol having an averagemolecular weight from 950 to 1,050, to one part of polyethylene glycol having an average molecular Weight from 285 to 315.

In preparing the drying and fixing aerosol compositions of the invention, it is beneficial first to prepare the suitable water-soluble mixture of one or more of each of the ordinarily liquid polyethylene glycols and the ordinarily solid polyethylene glycols. Such mixture then is dissolved in the selected alkanol, or mixture of alkanols, containing from about three to about ten percent of water based on the weight of aqueous alkanol, in the ratio of about to 60 grams of such polyethylene glycols mixture per liter of the aqueous alkanol solvent.

The resulting solution of the polyethylene glycols mixture in the water-containing alkanol conveniently can be called the fixingprotector solution. The fixing-protector solution then is mixed homogeneously with the selected aerosol propellant (either a single one or mixture of more than one) in such proportions for the final composition to contain from about 0.25 to about 0.92% of the mixture of the film-forming polyethylene glycols. In most cases, the fixing-protector solution and the selected propellant can be mixed in the proportion by weight of about one part of said solution to four parts of propellant, so long as the final composition will contain from about 0.25 to about 0.92% of the film-forming polyethylene glycols mixture.

The said fixing protector solution may include, for example, the polyethylene glycols in such proportions that an unconfined layer of a solution of their mixture in the alkanol solvent, containing up to about ten percent of water, contains about seven percent by weight of said glycols mixture.

The compositions of the invention are illustrated by, but not limited to, the following examples:

EXAMPLE 1 (i) Fifty grams of Carbowax 300" were admixed homogeneously in ten liters of commercial -91% isopropyl alcohol (balance by weight is water). Then two hundred grams of Carbowax 1000 similarly were admixed with this first solution. Finally, three hundred grams of Carbowax 4000 were added and uniformly mixed into that isopropyl alcohol solution of the others, to complete the preparation of the fixing-protector solution.

(ii) Sixteen liters of Freon-l2 were mixed with twenty-four liters of Freon-114 to provide a mixed propellant, having vapor pressure of 39.8 pounds per square inch at 70 F. The resulting forty liters of this mixed propellant was mixed with the ten liters of the fix ing-protector solution to give a total of :fifty liters of the complete drying and fixing aerosol composition. The latter then was loaded into four ounce aerosol dispensers in known manner.

The foregoing provides an advantageously effective formulation of the composition of the invention, containing 0.83% of dissolved polyethylene glycols, and about 16.8% of isopropanol. The vapor pressure of this complete composition is about thirty-five pounds per square inch at 70 F.

Other complete drying and fixing aerosol compositions are similarly prepared by adding suitable quantities of propellant to any of the following illustrative, but not intended to be restrictive, fixing-protector solutions:

EXAMPLE 2 Two hundred grams each of Carbowax 1000 and Carbowax 4000 were dissolved in ten liters of the ordinary ethyl alcohol.

EXAMPLE 3 One hundred grams of Carbowax 300 were uniformly admixed in ten liters of 90-91% isopropyl alcohol, into which solution there were uniformly dissolved two hundred grams each of Carbowax 1000 and Carbowax 4000.

EXAMPLE 4 One hundred grams of Carbowax 300 were dissolved in ten liters of ethyl alcohol 95%; and in the resulting solution there were successively dissolved three hundred grams of Carbowax 1540 and one hundred grams of Carbowax 4000.

EXAMPLE 5 One hundred and fifty grams of Carbowax 300 were dissolved in ten liters of 90-91% isopropyl alcohol, in which solution there were successively dissolved two hundred grams of Carbowax 1000 and two hundred and fifty grams of Carbowax 4000.

EXAMPLE 6 Eightyfive grams of Carbowax 300 were dissolved in ten liters of ethyl alcohol 95%. In the resulting solution there were successively dissolved two hundred and fifty grams each of Carbowax 1000 and Carbowax 4000.

EXAMPLE 7 One hundred and seventy-five grams of Carbowax 300 were dissolved in ten liters of 909l% isopropyl alcohol. In the resulting solution there were dissolved four hundred and thirty-five grams of Carbowax 4000.

EXAMPLE 8 Fifty grams of Carbowax 300 were dissolved in ten liters of 909l% isopropyl alcohol. Two hundred grams of Carbowax 1000 and three hundred grams of Carbowax 4000 then were dissolved in the solution prepared with the Carbowax 300.

The isopropyl alcohol of any of the Examples 1, 3, 5, 7 and 9 can be replaced in part or as a whole by the corresponding volume of any other herein applicable watermiscible lower alkanol (containing from about three to about ten percent of water), for example, ethyl alcohol 95%. Similarly, the ethyl alcohol of Examples 2, 4, 6 and 8 can be replaced in part or as a whole by the corresponding volume of any other herein mentioned lower alkanol (containing from about three to about ten percent of water), for example, 909l% isopropyl alcohol.

Each of the fixing-protector solutions of Examples 2 through 9 then conveniently is mixed with four times its volume of any aerosol propellant such as Freon-12, Freon-114, or suitable mixture of Freon-l2 with Freon-114 or Freon-ll, or other effective mixture of the type described herein and having an effective vapor pressure, for example, from about 30 to 75 pounds per square inch, at 70 F., or in any other proportion yielding a final complete composition containing from about 0.25 to about 0.92 percent by weight of film-forming polyethylene glycol content. The aerosol propellant of Example 1 similarly can be replaced in proportion and constitution so long as the polyethylene glycol content remains within this range.

When necessary in the preparation of any of the fixing protector solutions of any of the foregoing examples, or any of the above-described modifications of them or any others having any composition as broadly described herein, when the ambient temperature conditions make it advisable, to facilitate the solution of any of the ordinarily solid polyethylene glycols used, it can be liquified by heating it, for example, to 40-45 C. as for Carbowax 1000, or to 55-60 C. for Carbowax 4000, Carbowax 6000 or Carbowax 20M. In some such instances, sometimes it may be desirable also suitably to warm the alkanol solvent used.

The final drying and fixing aerosol compositions of the invention are used under the ordinary atmospheric conditions prevailing in any clinic, physicians ofiice, or clinical or research laboratory. After any individual cytological smear is applied to the ordinary glass microscope slide, the slide carrying the smear is held from about four to about six inches away from the nozzle of the aerosol dispenser containing the complete composition according to the invention. The distance of the slide from the nozzle need only be sufiicient for the spray emitted from it to avoid disturbing the cellular layout of the smear.

Generally it is necessary to expose the smear on the slide merely to one sweep of the spray emitted from the nozzle so held. As a precaution, the smear can be exposed to a second sweep of the spray. Each such slide carrying a thus sprayed smear merely needs to be set aside in the 8 ambient atmosphere of the room where it wa sprayed for it to be dry to the touch in from about five to about seven minutes.

The so dried slide (prepared by use of any composition of the various examples and any suitable modification of any thereof) shows a generally translucent, firm, nonbrittle, nonfiaking and fiow resistant, waxy film-coating over the smear and surface of the slide. In that condition, such slides can be mailed in any container merely designed to protect the thus coated smear from attrition and the glass slide from breakage, to any laboratory or other place where the slide is to be studied.

Moreover, as so coated any thus dried slides carrying such smears protected with a dry to the touch film-coating prepared from an aerosol composition of the invention, manifest fine cellular fixation and can be stored even for months with complete preservation of the original cell relationship and without any deterioration of the thus fixed cells of the smear.

The drying and fixing aerosol compositions of the invention can be used by the method of the invention for fixing and protecting cytological smears of any body exudation or fluid, for example, vaginal, bronchial, pleural, pericardial, and peritoneal fluids, or sputum. These aerosol compositions and the method are useful also for similarly treating the sediments of centrifuged body fluids such as pericardial, peritoneal, and pleural fluids.

When any of these so fixed and protected slides are received in the laboratory for staining preliminary to examination, they can be immersed directly in the aqueous hematoxylin staining solution bath. its water content dissolves away the Water-soluble polyethylene glycols filmcoating. Then as the thus originally protected dried and fixed smear is exposed, it is stained at the same time, just as any smear dried and fixed otherwise is exposed and immersed in an aqueous hematoxylin staining bath.

If desired, the dried and fixed, still polyethylene glycol film-coated smear, alternatively may be stained by the staining procedure described by Papanieolaou in Science, volume 95, pages 438439. In such case, prior to staining by his method, the polyethylene glycol filmcoated slide initially is dipped into water to dissolve away that film-coating. Such so stained slides are at least ontirely comparable with, and in many instances superior to, stained slides which initially are fixed and kept in a bath of a mixture of ethyl alcohol and ethyl ether.

Use of the drying and fixing aerosol compositions of the invention in the method of the invention thus gives strikingly advantageous ease and rapidity of fixation and with unusual economy of operation. It eliminates additional steps such as rehydration or supplemental fixation and other complications of prior procedures.

In addition, quite more important the compositions and method of the invention eliminate the cellular contamination prevalent in the repeated use of a common fixing solution maintained in a common fixing container. In the method of the invention instead each slide separately is covered only by its own directly received nebulizate of the drying and fixing aerosol solution emitted from the aerosol dispenser. The invention thus avoids any possibility for cells to be washed off from one slide and picked up on a succeeding slide bearing a smear taken from another source or subject.

While the invention has been explained in relation to certain illustrative embodiments of it, it is understood that many modifications and substitutions may be made in any of the specific embodiments within the scope of the appended claims which are intended also to cover equivalents of them.

We claim:

1. An aerosol composition composed of a mixture of water-soluble polyethylene glycols dissolved in an alkano! solvent, and a halo-alltane propellant, and which by weight consists essentially of:

(a) from about to about 19.5 percent of a watermiscible lower alkanol solvent which is a dehydrating agent and contains by weight from about 3 to about 10 percent of water;

(b) from about 0.25 to about 0.95 percent of the watersoluble polyethylene glycols mixture of at least one ordinarily liquid polyethylene glycol and at least one ordinarily solid polyethylene glycol so proportioned to one another that the film left after evaporation of said aqueous alkanol from an unconfined layer of a solution of from about to about 60 grams of said glycol mixture per liter of said aqueous alkanol deposited on a flat non-absorbent surface is dry to the touch, firm, non-brittle, non-flaky, flow-resistant and of waxy appearance;

(c) from about 89 to about 79 percent of a substantially non-toxic halo-alkane aerosol propellant chemically inert to said alkanol solvent and glycols mixture and miscible with its solution in said solvent, and having a molecular weight of from about 103 to about 204 and under 3 carbon atoms with only chlorine and fluorine linked to each carbon and at most only one hydrogen linked to the carbon when the alkane has only one carbon atom;

said water content of said alkanol solvent being suflicient to enable said solvent to dissolve the polyethylene glycols mixture content.

2. An aerosol composition as claimed in claim 1, wherein the propellant is at least one member of the class consisting of dichlorodifluoromethane, trichloromonofiuoromethane, dichloromonotluoromethane, monochlorodifluoromethanc; 1,2-dichlorotetrafluoroethane; and 1,1- dichloro-l-fluoro-2-chlorodifluoroethane; with the final composition having a vapor pressure of from about 30 to about 75 pounds per square inch at about 70 F.

3. An aerosol composition as claimed in claim 2, wherein the propellant is a mixture of dichlorodifluoromethane and l,2-dichlorotetrafluoroethane.

4. A composition as claimed in claim 3, wherein the dichlorodifluoromethane is about forty percent of the propellant.

5. An aerosol composition as claimed in claim 1, wherein the alkanol solvent is a member of the class consisting of isopropanol and ethanol.

6. A composition as claimed in claim 5, wherein the solvent is composed by weight of about 90 percent isopropanol and the balance is water.

7. An aerosol composition as claimed in claim 1, wherein the polyethylene glycol constituent is composed by weight of about 6 parts of polyethylene glycol of average molecular weight from 3,000 to 3,700, to 4 parts of polyethylene glycol of average molecular weight from 950 to 1,050, to one part of polyethylene glycol of average molecular weight from 285 to 315.

8. An aerosol compositionwhich consists essentially of an admixture by volume of:

(i) about four parts of a non-toxic halo-alkane aerosol propellant having a molecular weight of from about 103 to about 204 and under 3 carbon atoms with only chlorine and fluorine linked to each carbon and at most only one hydrogen linked to the carbon when the alkane has only one carbon atom; and

(ii) one part of a solution of (a) about 300 grams of polyethylene glycol of average molecular weight from 3000 to 3700, about 200 grams of polyethylene glycol of average molecular weight from 950 to 1050, and about 50 grams of polyethylene glycol of average molecular weight from 285 to 315, in (b) ten liters of 90 parts by weight of isopropanol and about 9 to 10 parts of water.

9. The method of fixing and drying a cytological smear on a suitable carrier that enables microscopically examining the smear, which method comprises:

(i) subjecting the smear as borne on such carrier to substantially overall exposed surface contact with the finely divided spray dispersion of the aerosol composition of claim 1, emitted from the nozzle of an aerosol dispenser held at such distance from the smear as to avoid disturbing its cellular layout by excessive force of the aerosol spray, to deposit over the smear a water-soluble covering layer of said polyethylene glycols; and

(ii) allowing the smear to remain exposed while there evaporates from it the aqueous alkanol therein along with any of the propellant contained in it and also the moisture from the smear within. a period of about seven minutes, thereby leaving the smear fixed and dry, free of contamination from content of any other smear, and covered with a water-soluble, dry to the touch, firm, non-brittle, non-flaking, flow-resistant, waxy coating of the mixed polyethylene glycols.

10. The method as claimed in claim 9, wherein said alkanol is a member of the class consisting of isopropanol and ethanol.

11. The method as claimed in claim 10, wherein the solvent is composed of about 90 percent isopropanol and its balance is water.

12. The method as claimed in claim 9, wherein the mixture of polyethylene glycols is composed of normally liquid and normally solid polyethylene glycols in such proportions that an unconfined layer of about a 7 percent solution of their mixture in the water-miscible alkanol solvent containing up to about 10 percent of water, when spread over a flat non-absorbent surface, after evaporation off of the aqueous alkanol, leaves said dry to the touch, firm, non-brittle, non-flaking, flow-resistant, waxy film.

13. The method as claimed in claim 12, wherein the mixture of polyethylene glycols is composed by weight of about 6 parts of polyethylene glycol of average molecular weight from 3,000 to 3,700, to 4 parts of polyethylene glycol of average molecular weight from 950 to 1,050, to one part of polyethylene glycol of average molecular weight from 285 to 315.

14. The method as claimed in claim 12, wherein the alkanol is a member of the class consisting of isopropanol and ethanol, and the propellant is at least one member of the class consisting of dichlorodifluorornethane, trichloromonofluoromethane, dichloromonofiuoromethane, monochlorodifluoromethane; 1,2-dichlorotetrafiuoroethane; and 1,1-dichloro-1-fluoro2-chlorodifluoroethane; with the final composition having a vapor pressure of from about 30 to about pounds per square inch at about 70 F.

15. The method as claimed in claim 14, wherein the propellant is about forty percent dichlorodifluoromethane and its balance is 1,2-dichlorotetrafiuoroethane.

16. The method as claimed in claim 15, wherein the fixing-protector solution is composed of about 300 grams of polyethylene glycol of average molecular weight from 3,000 to 3,700, about 200 grams of polyethylene glycol of average molecular weight from 950 to 1,050, and about 50 grams of polyethylene glycol of average molecular weight from 285 to 315, all dissolved in ten liters of parts by weight of isopropanol and about 9 to 10 parts by weight of water.

References Cited UNITED STATES PATENTS 8/1959 Nieburgs 167-845 Carbowax, Union Carbide Chem. Co., New York, 1958, pp. 7, 8, ll, 13, 14, 15, 24.

ALBERT T. MEYERS, Primary Examiner.

A. FAGELSON, Assistant Examiner. 

1. AN AEROSOL COMPOSITION COMPOSED OF A MIXTURE OF WATER-SOLUBLE POLYETHYLENE GLYCOLS DISSOLVED IN AN ALKANOL SOLVENT, AND A HALO-ALKANE PROPELLANT, AND WHICH BY WEIGHT CONSISTS ESSENTIALLY OF: (A) FROM ABOUT 10 TO ABOUT 19.5 PERCENT OF A WATERMISCIBLE LOWER ALKANOL SOLVENT WHICH IS A DEHYDRATING AGENT AND CONTAINS BY WEIGHT FROM ABOUT 3 TO ABOUT 10 PERCENT OF WATER; (B) FROM ABOUT 0.25 TO ABOUT 0.95 PERCENT OF THE WATERSOLUBLE POLYETHYLENE GLYCOLS MIXTURE OF AT LEAST ONE ORDINARILY LIQUID POLYETHYLENE GLYCOL AND AT LEAST ONE ORDINARILY SOLID POLYEHTYLENE GLYCOL SO PROPORTIONED TO ONE ANOTHER THAT THE FILM LEFT AFTER EVAPORATION OF SAID AQUEOUS ALKANOL FROM AN UNCONFINED LAYER OF A SOLUTION OF FROM ABOUT 15 TO ABOUT 60 GRAMS OF SAID GLYCOL MIXTURE PER LITER OF SAID AQUEOUS ALKANOL DEPOSITED ON A FLAT NON-ABSORBENT SURFACE IS DRY TO THE TOUCH, FIRM, NON-BRITTLE, NON-FLAKY, FLOW-RESISTANT AND OF WAXY APPEARANCE; (C) FROM ABOUT 89 TO ABOUT 79 PERCENT OF A SUBSTANTIALLY NON-TXIC HALO-ALKANE AEROSOL PROPELLANT CHEMICALLY INERT TO SAID ALKANOL SOLVENT AND GLYCOLS MIXTURE AND MISCIBLE WITH ITS SOLUTION IN SAID SOLVENT, AND HAVING A MOLECULAR WEIGHT OF FROM ABOUT 103 TO ABOUT 204 AND UNDER 3 CARBON ATOMS WITH ONLY CHLORINE AND FLUORINE LINKED TO EACH CARBON AND AT MOST ONLY ONE HYDROGEN LINKED TO THE CARBON WHEN THE ALKANE HAS ONLY ONE CARBON ATOM; SAID WATER CONTENT OF SAID ALKANOL SOLVENT BEING SUFFICIENT TO ENABLE SAID SOLVENT TO DISSOLVE THE POLYETHYLENE GLYCOLS MIXTURE CONTENT. 